Thyroid peroxidase (TPO), a thyroid cell surface protein, is the major autoantigen in autoimmune thyroid disease in humans. Understanding the precise molecular interaction between autoantibodies and T cells with TPO is a necessary step towards understanding the basis of the autoimmune response to TPO. We plan the following specific studies:- 1. TPO AUTOANTIBODY EPITOPES Polyclonal TPO autoantibodies in patients' sera are primarily directed to a restricted immunodominant region (IDR). We propose to identify many, if not all, TPO amino acid residues that comprise these epitopes using complementary approaches which, together, can help to complete the many pieces in the puzzle:- (i) Epitopic footprinting and identification of protected residues. Amino acids adjacent to these sentinel residues will then be identified by mutagenesis guided by the known three-dimensional structure of myeloperoxidase; (ii) X-ray diffraction crystals of TPO-TPO autoantibody complexes. 2. MOLECULAR CLONING OF NEW TPO AUTOANTIBODIES Human monoclonal autoantibodies are key reagents for defining B and T cell epitopes, as well as studying TPO processing and presentation to T cells. Autoantibodies cloned thus far are almost exclusively to the IDR. Important members of the TPO autoantibody repertoire remaining to be cloned include:- (i) Autoantibodies to the unique C-terminal region of the TPO molecule ectodomain with EGF-like and Sushi domains (amino acids 742-848); (ii) autoantibodies with dual thyroglobulin and TPO specificity ("TG-PO"). Important questions to be answered by these studies include:- (i) Are autoantibodies to the C-terminus the main component of serum autoantibodies with epitopes outside the IDR? (ii) Are some non-IDR TPO autoantibodies functionally important by inhibiting TPO enzymatic activity? 3. NATURALLY PROCESSED AND PRESENTED HUMAN T CELL EPITOPES FOR TPO Identification of TPO T cell epitopes is an important goal for understanding the autoimmune response to TPO. Identification of naturally processed peptide epitopes (NPPE) is preferable to testing T cell responsiveness with panels of synthetic peptides. We, therefore propose to extract TPO peptides from MHC molecules in APC and identify directly their amino acid sequence. We will use two different models:- (i) Human Epstein-Barr virus transformed human B cells that can capture, internalize and process TPO; (ii) Mouse fibroblasts co-expressing both human TPO and MHC class II molecules, whose injection induces TPO antibodies with the same epitopes as human autoantibodies. These studies will help answer the following questions:- (i) Which TPO T cell epitopes are most likely to be of pathophysiological importance? (ii) Does similarity between NPPE generated in different systems contribute to epitopic restriction of TPO autoantibodies? (iii) Conversely, do human autoantibodies influence the spectrum of NPPE generated from TPO.